The specific aims of this proposal address the broad question of whether variations in mitochondrial function can lead to variations in longevity. Several theories implicate the production of free radicals as a cause of aging. Mitochondria are the sites for a majority of free radical production in cells. Studies in humans and model organisms have suggested that naturally occurring variations in mitochondrial function may influence longevity. A few studies find that inheritance of longevity correlates more highly with maternal than paternal longevity. This suggests that mitochondrial DNA, which is inherited maternally, may influence longevity. Other studies indicate that damage caused in somatic tissue by the production of free radicals causes cellular components, particularly mitochondrial components, to become less efficient, which leads to more free radicals, more damage, and acceleration of the aging process. However, the results of many of these studies are clouded by variation in the genotypes of the test subjects. In this proposal Saccharomyces cerevisiae, an organism that is amenable to manipulation of both the nuclear and the mitochondrial genomes, will be used to address the question of mitochondrial function and longevity in two ways. (1) To test the hypothesis that germ-line mutations in mitochondrial DNA can lead to variations in life span, strains containing mitochondrial DNA mutations will be generated and the life spans of these strains will be assayed. Mitochondrial DNA will be selectively mutated using a novel method of mutagenesis and mitochondrial DNA transfer. Strains exhibiting slower growth rates will be selected for life span analyses. (2) To test the hypothesis that protection from oxidative damage positively correlates with extension of life span, strains containing mutations in the mitochondrial form of superoxide dismutase (SOD2) will be constructed and the life span of these strains will be assayed. Mitochondrial superoxide dismutase with altered activity will be isolated in E. coli sodA sodB after random mutagenesis using error-prone PCR. The altered forms of superoxide dismutase will be returned to yeast and expressed at normal levels under the control of the SOD2 promoter for life span analyses. Results from these experiments will serve to clarify the role of variations in mitochondrial function in determining longevity.